Use of cannabidiol in treatment of pulmonary hypertension

ABSTRACT

The present invention belongs to the field of medicine, and relates to use of cannabidiol (CBD) in the treatment of pulmonary arterial hypertension. The CBD can be used as the only active ingredient or combined with other active ingredients to prepare a medicament for treating pulmonary arterial hypertension. Specifically, the present invention relates to use of any one selected from (1) to (3) in the preparation of a medicament for treating and/or preventing pulmonary arterial hypertension: (1) CBD, (2) a plant extract containing the CBD; and preferably, the plant extract is a cannabis extract, and (3) a pharmaceutical composition containing the CBD and one or more pharmaceutically acceptable adjuvants. In the present invention, upon experimental researches, it has found that the CBD has the function of inhibiting pulmonary arterial hypertension, and is especially suitable for treating the pulmonary arterial hypertension caused by hypoxemia.

RELATED APPLICATIONS

This application is a United States National Stage Application filedunder 35 U.S.C 371 of PCT Patent Application Serial No.PCT/CN2017/093367, filed Jul. 18, 2017, the disclosure of all of whichare hereby incorporated by reference in their entirety.

FIELD

The present invention belongs to the field of medicine, and relates touse of cannabidiol in the treatment of pulmonary arterial hypertension.

BACKGROUND

Pulmonary arterial hypertension (PAH) has a complex etiology and iscaused by a variety of heart, lung, or pulmonary vascular diseases. ThePAH is pathologically characterized by pulmonary arteriolar vascularremodeling and pulmonary arterial vascular smooth muscle proliferation,is manifested as increased pulmonary circulation pressure andresistance, may result in an increased right cardiac load, right cardiacinsufficiency and decreased pulmonary blood flow, and thus causes aseries of clinical manifestations; and the PAH usually developsprogressively in the course of disease. In particular, right heartfailure caused by the increased pulmonary artery vascular tone is aserious threat to human life and health.

The PAH is divided into two categories: primary and secondary. With thegradual deepening of understanding of the PAH, the World HealthOrganization (WHO) classified PAH according to its etiology,pathophysiology, therapeutic regimen and prognosis in “PAH Conference”in 2003, and the American College of Chest Physicians (ACCP) and theEuropean Society of Cardiovascular Diseases (ESC) revised this in 2004.This classification method has guiding significance for the treatment ofPAH patients. It is generally believed that it is the PAH when the meanpulmonary artery pressure detected by a right cardiac catheter is ≥25mmHg in a calm state (Badesch et al., 2009). The PAH can be gradedaccording to the resting PAPm, with a mild level of 26-35 mm Hg; amoderate level of 36-45 mmHg; and a severe level of greater than 45mmHg.

The PAH mainly involves the pulmonary arteries and the right heart, andis manifested by right ventricular hypertrophy and right atrialdilatation. The main pulmonary artery is dilated and the surroundingpulmonary arterioles are sparse. Pulmonary arteriolar endothelial cellsand smooth muscle cells are proliferated and hypertrophied, the intimafibrosis is thickened, tunica media are hypertrophied, and the lumen isnarrow, occlusive, twisted and deformed and changes in clusters.Pulmonary venules can also have intimal fibrosis hyperplasia and lumenobstruction. Other manifestations of the PAH patient also includehypertrophy of pulmonary artery adventitias and veins, increasedexpression of TGF-β, and increased expression of matrix proteins such aselastin, fibronectin, cytochrome C and mucopolysaccharide.

The PAH is a disease that can be treated, but there is no effectivemethod for curing the PAH at present. Traditional treatment methodsinclude oxygen inhalation, cardiotonic, diuresis, calcium channelblockers and anticoagulant adjuvant therapeutic agents, which are mainlyused for relieving symptoms.

In recent years, the development and popularized use of targetedtherapeutic drugs (mainly including prostacyclin drugs, endothelinreceptor antagonists, phosphodiesterase-5 inhibitors and newly exploredsoluble guanylate cyclase agonists, 5-hydroxytryptamine transporterinhibitors, growth factor inhibitors, Rho kinase inhibitors, etc.), andtherapeutic methods such as living lung transplantation, and the likehave greatly improved the prognosis of the patient.

Although these drugs can relieve the symptoms of PAH to a certainextent, the PAH is still an incurable disease with high mortality. Themedian survival time of a patient that receives treatment is only 2.7years. Currently, there is still no specific cure method for the PAH.Therefore, it is particularly urgent to find a new specific therapeuticdrug.

SUMMARY

After in-depth research and creative work, the inventor has surprisinglyfound that cannabidiol can effectively inhibit pulmonary arterialhypertension (PAH) and is especially suitable for treating the PAHcaused by hypoxemia. Thus, the following invention is provided:

An aspect of the present invention relates to use of any one selectedfrom (1) to (3) in preparation of a medicament for treating and/orpreventing pulmonary arterial hypertension (PAH):

(1) cannabidiol, or a pharmaceutically acceptable salt or ester thereof,

(2) a plant extract containing cannabidiol; and preferably, the plantextract is a cannabis extract such as an industrial cannabis extract,and

(3) a pharmaceutical composition containing the cannabidiol or thepharmaceutically acceptable salt or ester thereof, and one or morepharmaceutically acceptable adjuvants.

For the use in one embodiment of the present invention, the PAH isprimary or secondary PAH; and preferably is arterial PAH (such asidiopathic PAH, heritable PAH, PAH caused by drugs and poisons orpersistent PAH of the newborn), PAH related to left heart diseases (suchas PAH caused by cardiac systolic dysfunction, PAH caused by diastolicdysfunction, or PAH caused by valvular diseases), PAH caused bypulmonary diseases (such as PAH caused by chronic obstructive pulmonarydiseases, PAH caused by emphysema or PAH caused by pulmonaryinterstitial diseases), PAH caused by hypoxemia (such as PAH caused bysleep apnea syndromes, or PAH caused by chronic altitude diseases suchas altitude heart diseases), or chronic thromboembolic PAH; and morepreferably is PAH caused by hypoxemia.

Cannabidiol (CBD) is one of cannabinoid substances, and its structuralformula is as shown in the following formula I:

The CBD, i.e., the compound of the formula I, can be purchasedcommercially (e.g., purchased from Sigma, etc.) or synthesized throughthe available technology by using commercially available raw materials.The synthesized CBD can be further purified by column chromatography,liquid-liquid extraction, molecular distillation or crystallization. Inaddition, the CBD can also be extracted from cannabis, especiallyindustrial cannabis.

In the present invention, upon experimental researches, it is foundthat, the cannabidiol and the aforementioned compound of the presentinvention have obvious therapeutic effects on animal models with PAHcaused by hypoxia and drug induction.

The pharmaceutically acceptable salt of the cannabidiol includes, butnot limited to, organic ammonium salts, alkali metal salts (sodiumsalts, and potassium salts), alkaline earth metal salts (magnesiumsalts, strontium salts, and calcium salts), etc.

In some embodiments of the present invention, the pharmaceuticallyacceptable salt of the cannabidiol may be a salt formed from thecannabidiol (CBD) with sodium hydroxide, potassium hydroxide, calciumhydroxide, magnesium hydroxide, aluminum hydroxide, lithium hydroxide,zinc hydroxide, barium hydroxide, ammonia, methylamine, dimethylamine,diethylamine, methylpyridine, ethanolamine, diethanolamine,triethanolamine, ethylenediamine, lysine, arginine, ornithine, choline,N,N′-diphenylmethylethylenediamine, chloroprocaine, diethanolamine,procaine, N-benzyl phenylethylamine, N-methylglucosamine piperazine,tris(hydroxymethyl)-aminomethane, etc.

In some embodiments of the present invention, the pharmaceuticallyacceptable ester of the cannabidiol may be a monoester of thecannabidiol and a C₀-C₆ alkyl carboxylic acid, or may be a diester ofthe cannabidiol and two identical or different C₀-C₆ alkyl carboxylicacids, the C₀-C₆ alkyl carboxylic acid may be a linear alkyl carboxylicacid, a branched alkyl carboxylic acid or a cycloalkyl carboxylic acid,such as, HCOOH, CH₃COOH, CH₃CH₂COOH, CH₃(CH₂)₂COOH, CH₃(CH₂)₃COOH,CH₃(CH₂)₄COOH, (CH₃)₂CHCOOH, (CH₃)₃CCOOH, (CH₃)₂CHCH₂COOH,(CH₃)₂CH(CH₂)₂COOH, (CH₃)₂CH(CH₃)CHCOOH, (CH₃)₃CCH₂COOH,CH₃CH₂(CH₃)₂CCOOH, cyclopropane carboxylic acid, cyclobutane carboxylicacid, and cyclopentane carboxylic acid.

The cannabis extract can be a cannabis, especially industrial cannabis,extract containing cannabidiol, such as an ethanol extracting solution,an ethanol extractum, etc. wherein the content of the cannabidiol is notparticularly limited, and the content of the cannabidiol in the cannabisextract can be further increased by means known to those skilled in theart, such as concentration, etc. In one embodiment of the presentinvention, the cannabis extract is an extractum, and preferably thecannabidiol content is 18%-25%.

Another aspect of the present invention relates to use of any oneselected from (1) to (3) in the preparation of a medicament forinhibiting inflammation, a medicament for inhibiting expression ofinflammatory factors, or a medicament for promoting expression of a genefor inhibiting inflammation (e.g., a Mgl₂ gene):

(1) cannabidiol, or a pharmaceutically acceptable salt or ester thereof,

(2) a plant extract containing cannabidiol; and preferably, the plantextract is a cannabis extract, and

(3) a pharmaceutical composition containing the cannabidiol or thepharmaceutically acceptable salt or ester thereof, and one or morepharmaceutically acceptable adjuvants.

For the use in one embodiment of the present invention, the inflammationis chronic low-grade inflammation.

For the use in one embodiment of the present invention, the inflammatoryfactor is selected from TNF-α and IL-6, and especially human TNF-α andhuman IL-6.

Another aspect of the present invention relates to a pharmaceuticalcomposition, including cannabidiol or a pharmaceutically acceptable saltor ester thereof, or including a plant extract (e.g., a cannabisextract) containing cannabidiol or a pharmaceutically acceptable salt orester thereof, and one or more pharmaceutically acceptable adjuvants.

In one embodiment of the present invention, cannabidiol is the onlyactive ingredient of the pharmaceutical composition. In anotherembodiment of the present invention, cannabidiol is used in combinationwith one or more other active ingredients known for preventing andtreating pulmonary arterial hypertension (PAH).

For the pharmaceutical composition in one embodiment of the presentinvention, the pharmaceutical composition further includes any one ormore selected from prostacyclin drugs, endothelin receptor antagonists,phosphodiesterase-5 inhibitors, soluble guanylate cyclase agonists,5-hydroxytryptamine transporter inhibitors, growth factor inhibitors,and Rho kinase inhibitors.

For the pharmaceutical composition in one embodiment of the presentinvention, wherein,

the prostacyclin drugs are selected from any one or more of Benapnost,Treprostinil, iloprost and Ventavis;

the endothelin receptor antagonist is bosentan; and/or

the phosphodiesterase-5 inhibitor is selected from Sildenafil,Vardenafil and Tadalafil.

The preparation form of the pharmaceutical composition can be anypharmaceutical dosage form, and the dosage forms include tablets,sugar-coated tablets, film-coated tablets, enteric-coated tablets,capsules, hard capsules, soft capsules, oral liquids, buccal agents,granules, dissolved medicines, pills, pulvis, pastes, sublimedpreparations, suspensions, powders, solutions, injections,suppositories, ointments, plasters, creams, sprays, drops and patches;and preferably oral dosage forms, such as capsules, tablets, oralliquids, granules, pills, pulvis, sublimed preparations, pastes, etc.The oral dosage form may contain common excipients, such as a binder, afiller, a diluent, a tableting agent, a lubricant, a disintegrant, acolorant, a flavoring agent and a humectant, and the tablets may becoated if necessary. Suitable fillers include cellulose, mannitol,lactose and other similar fillers; suitable disintegrants includestarch, polyvinylpyrrolidone, and starch derivatives such as sodiumstarch glycolate; and suitable lubricants include, for example,magnesium stearate. Suitable pharmaceutically acceptable humectantsinclude sodium dodecyl sulfate.

Preferably, the pharmaceutical composition is an oral preparation. Upontests the inventor has found that, the mediciment prepared from thecannabidiol has an obvious therapeutic effect on a mouse suffering fromPAH through oral gavage.

The treatment and/or prevention effective dosage of the pharmaceuticalcomposition of the present invention is 0.1-200 mg/kg body weight/day.The effective dosage of the pharmaceutical composition of the presentinvention is preferably 0.1-100 mg/kg body weight/day; and morepreferably 0.1-50 mg/kg body weight/day. It is recommended that theeffective dosage of the pharmaceutical composition in the human body ispreferably 0.1-50 mg/kg body weight/day; and more preferably 0.5-30mg/kg body weight/day. The “effective dosage for treatment and/orprevention” can be used for single drug administration or combined drugadministration for related diseases.

The preferred use method of the pharmaceutical composition (medicament)for treating pulmonary arterial hypertension is oral administration, andthe preferred dosage is 0.5-30 mg/kg daily.

Another aspect of the present invention relates to a combined product,including a product 1 and a product 2, wherein:

the product 1 is any one selected from (1) to (3):

(1) cannabidiol, or a pharmaceutically acceptable salt or ester thereof,

(2) a plant extract containing cannabidiol; and preferably, the plantextract is a cannabis extract, and

(3) a pharmaceutical composition containing the cannabidiol or thepharmaceutically acceptable salt or ester thereof, and one or morepharmaceutically acceptable adjuvants;

the product 2 is any one or more selected from prostacyclin drugs,endothelin receptor antagonists, phosphodiesterase-5 inhibitors, solubleguanylate cyclase agonists, 5-hydroxytryptamine transporter inhibitors,growth factor inhibitors and Rho kinase inhibitors;

and the products 1 and 2 are each individually packaged;

preferably, both products 1 and 2 are in the form of oral preparations.

For the combined product in one embodiment of the present invention,wherein

the prostacyclin drugs are any one or more selected from Benapnost,Treprostinil, iloprost and Ventavis;

the endothelin receptor antagonist is bosentan; and/or

the phosphodiesterase-5 inhibitor is selected from Sildenafil,Vardenafil and Tadalafil.

A further aspect of the present invention relates to a product selectedfrom any one of (1) to (3), used for treating and/or preventingpulmonary arterial hypertension (PAH):

(1) cannabidiol, or a pharmaceutically acceptable salt or ester thereof,

(2) a plant extract containing cannabidiol; and preferably, the plantextract is a cannabis extract, and

(3) a pharmaceutical composition containing the cannabidiol or thepharmaceutically acceptable salt or ester thereof, and one or morepharmaceutically acceptable adjuvants.

For the product in one embodiment of the present invention, the PAH isprimary or secondary PAH; and preferably is arterial PAH (such asidiopathic PAH, heritable PAH, PAH caused by drugs and poisons orpersistent PAH of the newborn), PAH related to left heart diseases (suchas PAH caused by cardiac systolic dysfunction, PAH caused by diastolicdysfunction, or PAH caused by valvular diseases), PAH caused bypulmonary diseases (such as PAH caused by chronic obstructive pulmonarydiseases, PAH caused by emphysema or PAH caused by pulmonaryinterstitial diseases), PAH caused by hypoxemia (such as PAH caused bysleep apnea syndromes, or PAH caused by chronic altitude diseases suchas altitude heart diseases), or chronic thromboembolic PAH; and morepreferably is PAH caused by hypoxemia.

A further aspect of the present invention relates to a product selectedfrom any one of (1) to (3), used for inhibiting inflammation, inhibitingexpression of inflammatory factors, or promoting expression of a genefor inhibiting inflammation (e.g., a Mgl₂ gene):

(1) cannabidiol, or a pharmaceutically acceptable salt or ester thereof,

(2) a plant extract containing cannabidiol; and preferably, the plantextract is a cannabis extract, and

(3) a pharmaceutical composition containing the cannabidiol or thepharmaceutically acceptable salt or ester thereof, and one or morepharmaceutically acceptable adjuvants.

For the product in one embodiment of the present invention, theinflammatory factor is selected from TNF-α and IL-6, and especiallyhuman TNF-α and human IL-6.

Another aspect of the present invention relates to a method of treatingand/or preventing pulmonary arterial hypertension, including the step ofadministering an effective amount of a product selected from any one of(1) to (3) to a subject in need:

(1) cannabidiol, or a pharmaceutically acceptable salt or ester thereof,

(2) a plant extract containing cannabidiol; and preferably, the plantextract is a cannabis extract, and

(3) a pharmaceutical composition containing the cannabidiol or thepharmaceutically acceptable salt or ester thereof, and one or morepharmaceutically acceptable adjuvants.

For the method in one embodiment of the present invention, the subjectis a mammal, and especially human beings.

For the method in one embodiment of the present invention, the PAH isprimary or secondary PAH; and preferably is arterial PAH (such asidiopathic PAH, heritable PAH, PAH caused by drugs and poisons orpersistent PAH of the newborn), PAH related to left heart diseases (suchas PAH caused by cardiac systolic dysfunction, PAH caused by diastolicdysfunction, or PAH caused by valvular diseases), PAH caused bypulmonary diseases (such as PAH caused by chronic obstructive pulmonarydiseases, PAH caused by emphysema or PAH caused by pulmonaryinterstitial diseases), PAH caused by hypoxemia (such as PAH caused bysleep apnea syndromes, or PAH caused by chronic altitude diseases suchas altitude heart diseases), or chronic thromboembolic PAH; and morepreferably is PAH caused by hypoxemia.

A further aspect of the present invention relates to a method ofinhibiting inflammation, inhibiting expression of inflammatory factorsor promoting expression of a gene for inhibiting inflammation (e.g., aMgl₂ gene), including the step of administering an effective amount of aproduct selected from any one of (1) to (3) to a subject in need:

(1) cannabidiol, or a pharmaceutically acceptable salt or ester thereof,

(2) a plant extract containing cannabidiol; and preferably, the plantextract is a cannabis extract, and

(3) a pharmaceutical composition containing the cannabidiol or thepharmaceutically acceptable salt or ester thereof, and one or morepharmaceutically acceptable adjuvants.

For the method in one embodiment of the present invention, theinflammation is chronic low-grade inflammation.

For the method in one embodiment of the present invention, theinflammatory factor is selected from TNF-α and IL-6.

It should be pointed out that, the dosage and use method of the activeingredient cannabidiol depend on many factors, including the age, bodyweight, gender, natural health status, and nutritional status of thepatient, the activity intensity of the compound, the administrationtime, the metabolic rate, the severity of the disease and the subjectivejudgment of the physician. The dosage (effective amount) of the usedcannabidiol is preferably 0.1-50 mg/kg body weight/day, more preferably0.5 mg/kg-30 mg/kg body weight/day, 0.5 mg/kg-20 mg/kg body weight/day,5 mg/kg-30 mg/kg body weight/day or 5 mg/kg-20 mg/kg body weight/day,further more preferably 0.5 mg/kg-10 mg/kg body weight/day, andparticularly preferably 0.5 mg/kg-5 mg/kg body weight/day. Preferably,the administering is performed orally.

In the present invention, the term “effective amount” refers to a dosethat can achieve the treatment, prevention, alleviation and/or remissionof the disease or condition of the present invention in a subject.

The term “subject” may refer to a patient or other animals, especiallymammals, such as human, a dog, a monkey, a cow, a horse, etc., thatreceives/receive the composition of the present invention to treat,prevent, alleviate, and/or relieve the disease or condition of thepresent invention.

The term “disease and/or condition” refers to a physical state of thesubject, which is related to the disease and/or condition of the presentinvention.

In the present invention, the term “pulmonary arterial hypertension” andthe commonly referred hypertension are two distinctly differentdiseases. A heart of human is divided into a left heart system and aright heart system. What we usually call hypertension refers to thepressure rise in arteries started from the left heart system forsupplying blood to the whole body; and the arteries started from theright heart system are specially responsible for supplying blood to thelung, and are called pulmonary arteries, and the pressure rise in sucharteries is called pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a disease or pathophysiologicalsyndrome caused by abnormal pressure rise in pulmonary artery due toknown or unknown reasons. The pulmonary circulation disorder and rightheart overload exist, leading to failure and even death of the rightheart, and in the later stage of PAH patients, hypotension may develophypotension due to cardiac ejection dysfunction.

The term “hypertension” refers to a clinical syndrome mainlycharacterized by an increase in systemic arterial blood pressure(systolic and/or diastolic pressure) (the systolic pressure ≥140 mm Hg,and the diastolic pressure ≥90 mm Hg), which may be accompanied byfunctional or organic damage to organs such as the heart, brain andkidney.

The term “TNF-α” refers to the tumor necrosis factor-α, which is acytokine involved in systemic inflammation, meanwhile is also one ofmany cytokines that cause acute phase reactions, and is mainly secretedby macrophages. The main function of the tumor necrosis factor α is toregulate the function of immune cells. In the present invention, unlessotherwise specified, TNF-α refers to a protein as shown by the GenBankaccession number: NP_038721.1, or a fragment thereof having the functionof the TNF-α. In one embodiment of the present invention, the nucleicacid sequence of the TNF-α gene is as shown in Gene ID: 21926 or itsdegenerate sequence.

The term “IL-6” refers to interleukin 6, which is a cytokine and belongsto a one of interleukins. IL-6 can be produced by fibroblasts,monocytes/macrophages, T lymphocytes, B lymphocytes, epithelial cells,keratinocytes or various tumor cells. Interleukin 6 can stimulate theproliferation, and differentiation of cells involved in immune response,and improve the function of the cells. In the present invention, unlessotherwise specified, IL-6 refers to a protein as shown by the GenBankaccession number: NP_001300983.1, or a fragment thereof having thefunction of IL-6. In one embodiment of the present invention, thenucleic acid sequence of the IL-6 gene is as shown in Gene ID: 16193 orits degenerate sequence.

The term “Mgl₂” refers to macrophage galactose N-acetyl-galactosaminespecific lectin 2. In the present invention, unless otherwise specified,Mgl₂ refers to a protein as shown by the GenBank accession number:NP_660119.1, or a fragment thereof having the function of Mgl₂. In oneembodiment of the present invention, the nucleic acid sequence of theMgl₂ gene is as shown in Gene ID: 216864 or its degenerate sequence.

In the present invention, unless otherwise specified, the products 1 and2 are only for clear reference, and do not have the meaning of order.

In the present invention, unless otherwise specified, the cannabis ispreferably industrial cannabis; and the cannabis extract is preferablyan industrial cannabis extract.

Beneficial Effects of the Invention

In the invention, upon experimental researches, the effect ofcannabidiol on inhibiting pulmonary arterial hypertension has beenfound, showing that cannabidiol has a good therapeutic effect onpulmonary arterial hypertension, and is especially suitable for treatingpulmonary arterial hypertension caused by hypoxemia.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 : CBD reduces the increased right ventricular systolic pressureas caused by hypoxia, N=10/group, the numeral value is the averagevalue±standard error, and when the wild-type normoxia group is taken asa reference, ***P<0.001; and when the wild-type hypoxia group is takenas a reference, ###P<0.001.

FIG. 2 : CBD reduces right ventricular hypertrophy caused by hypoxia,N=10/group, the numeral value is the average value±standard error, andwhen the wild-type normoxia group is taken as a reference, ***P<0.001;and when the wild-type hypoxia group is taken as a reference,###P<0.001.

FIG. 3 : results of pulmonary arteriolar vascular HE staining (FIGS.3A-3D) and elastic fiber staining (FIGS. 3E-3H), wherein the samples ofFIGS. 3A-3D are paraffin sections of lung tissues of mice from groups1-4 in sequence, and the samples of FIGS. 3E-3H are paraffin sections oflung tissues of mice from groups 1-4 in sequence.

FIG. 4 : CBD reduces the vascular remodeling rate caused by hypoxia,N=10/group, the numeral value is the average value±standard error, andwhen the wild-type normoxia group is taken as a reference, ***P<0.001;and when the wild-type hypoxia group is taken as a reference, ###P<0.01.

FIG. 5 : CBD inhibits LPS-induced activation of primary alveolarmacrophages in vitro, The numerical value is the average value±standarderror, and when the negative control group is taken as a reference,*P<0.05, **P<0.01; and when the LPS treatment group is taken as areference, #P<0.05, ##P<0.01.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Hereinafter, embodiments of the present invention will be described indetail with reference to examples, but those skilled in the art willunderstand that the following examples are only for illustrating thepresent invention and should not be considered as limiting the scope ofthe present invention. If no specific conditions are specified in theexamples, the embodiments will be carried out according to conventionalconditions or the conditions recommended by the manufacturer. All of theused agents or instruments which are not specified with the manufacturerare conventional commercially-available products.

Example 1: Test on Effect of Cannabidiol on Pulmonary ArterialHypertension Caused by Hypoxia

1. Experimental Animals, Reagents and Instruments

(1) Experimental Animals

Healthy and lively C57BL/6 mice at the age of 4-6 weeks, with glossyhair colors, and body weights of (25.15±2.15) g (Laboratory AnimalCenter of Academy of Military Medical Sciences, SPF grade). The micewere divided into four groups:

Group 1 (wild-type normoxia, used as a control group): 10 female miceand 10 male mice under normoxia conditions;

Group 2 (wild-type hypoxia, used as a control group): 10 female mice and10 male mice under hypoxia conditions;

Group 3 (10 mg/kg, used as an experimental group): 10 female mice and 10male mice were treated with 10 mg/kg by gavage under hypoxia conditions;

Group 4 (50 mg/kg, used as an experimental group): 10 female mice and 10male mice were treated with 50 mg/kg by gavage under hypoxia conditions.

(2) Administration Method

The mice of groups 2-4 were placed in an animal feeding chamber undernormal pressure and hypoxia, the oxygen concentration in the chamber wasmaintained at 9%-11%, and the temperature in the chamber was maintainedat 22-26° C. The mice of group 1 inhaled normal atmosphere air, and theother conditions were the same as those for mice of groups 2-4.

Previous research results showed that a pulmonary arterial hypertensionmodel using the significant increase in right ventricular systolicpressure as a standard could be established after 14 days of continuoushypoxia in mice (Ricard, N., Tu, L., Le Hiress, M., Huertas, A., Phan,C., Thuillet, R., Sattler, C., Fadel, E., Seferian, A., Montani, D., etal. (2014). Increased pericyte coverage mediated by endothelial-derivedfibroblast growth factor-2 and interleukin-6 is a source of smoothmuscle-like cells in pulmonary hypertension. Circulation 129,1586-1597.). In order to explore whether CBD has a therapeutic effect onpulmonary arterial hypertension or not, the inventor first treated micefor 14 days in a continuous hypoxia environment to establish a model ofpulmonary arterial hypertension (the groups 2-4), and thenadministration was started from day 15 to day 21 through gavage once aday, the mice were treated every day for 7 days (the group 3-4), anddetection was performed on day 21.

The specific detection steps were described in Part 2 “ExperimentalMethod” hereafter.

(3) Experimental Reagents

Sodium pentobarbital (sigma); cannabidiol (Yunnan Hansu BiotechnologyCo., Ltd); Crystal Violet, Victoria Blue B (Sinopharm Chemical ReagentCo., Ltd.); New fuchsine (Tokyo Chemical Industry).

(4) Experimental Instruments

A multi-channel physiological recorder (BE-EH4) from B&E TEKSYSTEMS LTD;an OLYMPUS microscope (CX4) from Olympus (China) Co., Ltd. (OCN); ahypoxia box (CJ-DO2) from Changsha Changjin Technology Co., Ltd; afluorescence quantitative PCR instrument (Light Cycler480II) from RocheApplied Science.

2. Experimental Method

(1) Determination of Right Ventricular Systolic Pressure (RVSP)

On day 21, the mice were anesthetized by intraperitoneal injection ofsodium pentobarbital (35 mg/kg), and the right ventricular systolicpressure was measured by using a catheter matched with a physiologicalinstrument with reference to the right-heart catheterization reported bySong et al. (Song, Y., Jones, J. E., Beppu, H., Keaney, J. F., Jr.,Loscalzo, J., and Zhang, Y. Y. (2005). Increased susceptibility topulmonary hypertension in heterozygous BMPR2-mutant mice. Circulation112, 553-562.). A tip of the catheter was connected with a signalacquisition and processing system of the multi-channel physiologicalrecorder, and the position of the tip of the catheter was judgedaccording to the blood pressure values and the migration changes ofwaveforms of the pressure curve displayed by a monitor. After thecatheter entered the right ventricle, RVSP was determined and recorded.

(2) Measurement of Right Ventricular Hypertrophy (RVH) Index

According to the method by Ryan et al., after anesthesia of a mouse, thethoracic cavity of the mouse was opened to pick the heart, all bloodvessels and ventricles were stripped off from the heart, the rightventricle was cut off, the weight of the right ventricle and the weightsof the left ventricle plus diaphragm were weighed respectively, and theweight of the right ventricle was divided by the combined weight of theleft ventricle and diaphragm (RV/(LV+S)).

(3) HE Staining

Sample: paraffin sections of mouse lung tissue.

Objective: observing the pathology of lung tissues to detect whetherthere is a vascular wall thickening phenomenon or not.

The cut paraffin sections were placed into an oven at 55° C. for 10 min.

1) the paraffin sections were dewaxed and went downward to 70% ethanol;

xylene I: 15 min

xylene II: 7 min

1:1 toluene-ethanol solution: 5 min

Ethanol of various stages: each for 5 min

2) staining with a hematoxylin solution for 10-15 min;

3) rinsing with tap water for 2 min;

4) differentiation in a 0.5% hydrochloric acid-alcohol solution for 8 s;

5) turning back to blue with tap water for 10 min;

6) 70% alcohol→80% alcohol, each for 2 min;

7) 0.5% eosin-alcohol dye liquor for 50-70 s;

8) color separation with 90% ethanol and 95% ethanol, each for 3 min;

9) dehydrating with absolute ethanol I and II, each for 3 min;

10) absolute ethanol:xylene (1:1) for 3 min;

11) xylene I for 3 min;

12) xylene II for 3 min;

Mounting with a neutral gum;

After the completion of staining, the nucleus was presented asblue-purple, and the cytoplasm was presented as pink.

(4) Elastic Fiber Staining (Elastic staining)

Sample: paraffin sections of mouse lung tissue.

Preparation of dye liquor: 1 g of Victorian Blue B.

1 g of New Fuchsin, and 1 g of Crystal Violet.

The materials were dissolved in 200 ml of hot water, 4 g of resorcinol,4 g of dextrin and 50 ml of 30% ferric chloride (prepared immediatelybefore use) were sequentially added, the mixture was boiled for 5 minand filtered, the precipitate and the filter paper were dissolved in 200ml of 95% ethanol, boiled for 15 min-20 min and filtered (in a waterbath), and supplemented with 95% ethanol to 200 ml, and finally 2 ml ofconcentrated hydrochloric acid was added. The dye liquor was sealed andstored in dark place.

Staining Method

xylene I: 10 min; xylene II: 10 min; 100% ethanol: 5 min; 90% ethanol: 5min; tap water: 5 min; 0.5% potassium permanganate for 5 min; rinsingwith tap water for 2-3 min; a 1% oxalic acid solution for 2-3 min (justfor bleaching); rinsing with tap water for 2-3 min; 95% ethanol for 2-3min; staining with an Elastic dye liquor for 2 h; washing the dye liquoraway with 95% ethanol; rinsing with tap water for 2-3 min; staining witha Van Gieson dye liquor for 1 min; dehydrating rapidly: 80% ethanol for1 min, 90% ethanol for 1 min, absolute ethanol I for 5 min, absoluteethanol II for 5 min, xylene I for 5 min, and xylene II for 5 min.

(5) Vascular Remodeling Rate

The method by Keegan et al. (Keegan, A., Morecroft, I., Smillie, D.,Hicks, M. N., and MacLean, M. R. (2001). Contribution of the 5-HT(1B)receptor to hypoxia-induced pulmonary hypertension: converging evidenceusing 5-HT(1B)-receptor knockout mice and the 5-HT(1B/1D)-receptorantagonist GR127935. Circulation research 89, 1231-1239) was employed,wherein counting was conducted with lung paraffin sections that hadpreviously stained for elastic fibers, and 50-100 μm of pulmonaryarterioles far away from the central airway were selected for counting,and a blood vessel that had a remodeling part exceeding ½ or more of thecircumference of the blood vessel was recorded as a remodeled bloodvessel.

(6) Statistical Treatment

The measurement data were expressed as mean±standard error, andstatistical processing was conducted by employing SPSS 22.0. Thestatistical tests were all based on a double-tailed T test.

3. Experimental Results

(1) Right Ventricular Systolic Pressure in PAH Mice

The results were as shown in FIG. 1 .

After 21 days of continuous hypoxia, the mean right ventricular systolicpressure in the hypoxia model control group was (25.55±2.29) mmHg, whichwas significantly higher than that in the normoxia control group(17.54±1.48) mmHg, with a statistically significant difference(P<0.001).

After the CBD treatment, the mean right ventricular systolic pressure inthe 10 mg/kg experimental group was (16.90±2.31) mmHg, which wassignificantly lower than that in the hypoxia model control group, with astatistically significant difference (P<0.001); the mean rightventricular systolic pressure in the 50 mg/kg experimental group was(17.92±2.37) mmHg, which was also significantly lower than that in themodel group, but there was no significant difference in the results ofthe two dose treatment groups (10 mg/kg and 50 mg/kg).

(2) The CBD Treatment Significantly Inhibited the Right VentricularHypertrophy Index of the PAH Mice

The results were as shown in FIG. 2 .

The right ventricular hypertrophy index of the mice in the model groupwas (32.62±1.41)%, which was significantly higher than that of the micein the normoxia control group (25.99±1.17)%. After the CBD treatment,the right heart index of the mice in the 10 mg/kg and 50 mg/kgexperimental groups were (30.18±1.01)% and (29.90±1.19)%, respectively,which were significantly lower than that of the model group, with astatistically significant difference (P<0.001), but there was nosignificant difference between the different dose treatment groups.

(3) Pathological Changes of Pulmonary Arterioles

The results were as shown in FIGS. 3A-3H and 4 .

In the hypoxia model group, the vascular wall of the mice wassignificantly thickened and remodeled. After the CBD treatment, thepulmonary arteriole remodeling was significantly reduced, and theremodeling rate was significantly reduced.

Both dose groups of cannabidiol could reduce the right ventricularsystolic pressure and inhibit the right ventricular (RV) hypertrophyindex; and the pathological remodeling had an improvement effect,including the reduction of arterial medial wall thickness ratio, thereduction of vascular wall medial cross-sectional area ratio and thereduction of the right ventricular hypertrophy.

Example 2: In Vitro Experiment of Treating LPS-Induced Macrophages withCannabidiol

1. Experimental Animals, Reagents and Instruments

2-month-old C57BL/6 mice (Laboratory Animal Center of Academy ofMilitary Medical Sciences, SPF grade)

RPMI-1640 (Sigma)

Cannabidiol (CBD, Yunnan Hansu Biotechnology Co., Ltd)

LPS (Sigma)

Fluorescence quantitative PCR instrument (Roche)

2. Experimental Method

Primary alveolar macrophages were isolated according to the method byYang et al. (Yang, H. M., Ma, J. Y., Castranova, V., and Ma, J. K.(1997). Effects of diesel exhaust particles on the release ofinterleukin-1 and tumor necrosis factor-alpha from rat alveolarmacrophages. Experimental lung research 23, 269-284.): two-month-oldmice were taken and injected intraperitoneally with pentobarbital to beanesthetized. The mice were fixed at a supine position, and disinfectedat the neck, and the neck skin was cut open to strip gland muscles andexpose the trachea. A small opening was cut in an upper part of thetrachea without cutting off. A small white pipette tip was inserted ontoa 1 ml pipette to pipette 1 ml PBS at 4° C. (free of Ca²⁺, free of Mg²⁺,0.6 mm EDTA) [50 ml PBS+0.00876 g EDTA] into the lung from the opening,and then pipetted out from the opening after blowing in. Then fresh PBSwas taken and pipetted into and out from the opening repeatedly for 3-4times. The collected alveolar irrigation solution was centrifuged at200× g for 5 minutes, and the supernatant was discarded. The cells werewashed once with a serum-free RPMI-1640 culture medium and centrifugedat 200× g for 5 minutes. The cells were resuspended by adding aserum-containing RPMI-1640 and then inoculated. The cells were subjectedto adherence for 2 h, and then washed for three times with PBS. Aserum-containing RPMI-1640 was added, and the cells were cultured under5% carbon dioxide at 37° C.

Alveolar macrophages of the mice were taken and randomly divided intosix treatment groups (about 5×10⁶ cells in each group), wherein thefirst group was a negative control; the second group was induced by 100ng/mL LPS (LPS was added into the culture medium) to simulate ainflammatory reaction model in vivo; the third group was a CBD controlgroup (5 μM CBD was added into the culture medium); and the fourth groupwas pre-treated with 0.5 μM CBD for 0.5 h, and then induced by 100 ng/mLLPS (0.5 μM CBD and 100 ng/mL LPS were added into the culture medium).The fifth group was pre-treated with 1 μM CBD for 0.5 h and then inducedby 100 ng/mL LPS (1 μM CBD and 100 ng/mL LPS were added into the culturemedium). The sixth group was pre-treated with 5 μM CBD for 0.5 h andthen induced by 100 ng/mL LPS (5 μM CBD and 100 ng/mL LPS were addedinto the culture medium). The cells were collected after treatment for 5h, and the expression changes of cellular inflammation cytokines TNF-α,IL-6 and Mgl₂ in each group were detected by the fluorescencequantitative PCR instrument.

Primers for detecting TNF-α (SEQ ID NO: 1)forward primer: CCCTCACACTCAGATCATCTTCT (SEQ ID NO: 2)reverse primer: GCTACGACGTGGGCTACAG Primers for detecting IL-6:(SEQ ID NO: 3) forward primer: GAGGATACCACTCCCAACAGACC (SEQ ID NO: 4)reverse primer: AAGTGCATCGTTGTTCATACA Primers for detecting Mg1₂:(SEQ ID NO: 5) forward primer: AGGCAGCTGCTATTGGTTCTCTGA (SEQ ID NO: 6)reverse primer: AGTTGACCACCACCAGGTGAGAAT

3. Experimental Results

The results were as shown in FIG. 5 .

The results showed that CBD could significantly inhibit the expressionof LPS-induced inflammation-related TNF-α and IL-6 in a dose-dependentmanner, and promote the expression of Mgl₂, a gene that inhibitedinflammation.

Although specific embodiments of the present invention have beendescribed in detail, those skilled in the art will understand thefollowing things. In light of all the teachings that have beendisclosed, various modifications and replacements can be made to thosedetails, which are all within the protection scope of the presentinvention. The full scope of the present invention is given by theappended claims and any equivalents thereof.

The invention claimed is:
 1. A method of treating pulmonary arterialhypertension in a human in need thereof consisting essentially ofadministering 0.1 mg/kg body weight-100 mg/kg body weight of syntheticcannabidiol to effectively treat the pulmonary arterial hypertension inthe human in need thereof.